Breast cancer continues to be a leading cause of cancer related deaths among women in the United States. The long-term goal of this research project is to develop a safe and effective strategy for prevention of breast cancer in women using a novel Ayurvedic medicine constituent- Withaferin-A (WA). This preclinical research project stems from our published and preliminary unpublished results demonstrating that WA treatment decreases viability of cultured breast cancer cells by causing apoptosis and the proapoptotic effect of WA correlates with generation of reactive oxygen species (ROS), activation of c-Jun- N-terminal kinases (JNK1/2), and induction of proapoptotic proteins Bax, Bak, and Bim. Noticeably, WA administration significantly retards growth of MDA-MB-231 human breast cancer xenografts in female nude mice without causing any harmful side effects. Despite these promising results, however, significant gaps exist in our understanding of the mechanism(s) of WA-induced apoptosis. For example, the precise mechanism of WA-mediated ROS production remains elusive. Likewise, the signaling pathways downstream of ROS production and JNK1/2 activation in execution of WA-induced apoptosis are unclear. Studies proposed in this application will not only fill these mechanistic gaps in our knowledge but also determine in vivo efficacy of WA for prevention of breast cancer in a transgenic mouse model. Based on the results of our preliminary studies, we hypothesize that WA treatment selectively causes ROS/JNK-mediated apoptosis in breast cancer cells leading to chemoprevention of mammary carcinogenesis. Specific Aims: The specific aims of this project are to: (1) Determine the mechanism of WA-mediated ROS production using MDA-MB-231, MCF-7, BRI-JM04, and MCF-10A cell lines as a model; (2) Determine the mechanism of WA-mediated activation of JNK1/2 using the above mentioned cell lines as a model; (3) Determine the signaling pathways downstream of ROS production and JNK1/2 activation in execution of WA- induced apoptosis using above cell lines; the MCF-10A cell line is included in specific aims 1-3 to gain insight into the mechanism of relative resistance of normal mammary epithelial cells towards WA-induced apoptosis; (4) Determine the efficacy of dietary WA administration for prevention of mammary carcinogenesis using MMTV-neu mice; and (5) Determine the mechanism by which dietary WA may prevent breast cancer development in MMTV-neu mice using mammary tumor tissues from control and WA-treated MMTV-neu mice. Significance of the Proposed Research: Positive outcome of the proposed preclinical in vivo efficacy study will provide impetus for clinical trials to determine chemopreventive effectiveness of WA against human breast cancer. The value of defining the mechanism of anticancer effect of WA may be realized in a variety of ways including identification of biomarker(s) of WA response potentially useful in future clinical trials and optimization of WA-based chemopreventive regimens against breast cancer.